Pulmonary epithelium is divided into three histopathologically and biologically distinct compartments: bronchi, bronchioli, and alveoli. Two of the main types of human lung cancers (squamous cell and small cell carcinoma) are typically centrally located and originate from bronchi. In contrast, the most common lung cancer type adenocarcinoma is typically a peripheral tumor which originates from the peripheral airway cells composed of bronchiolar Clara cells or alveolar type 2 cells. Our long term goal is to characterize the morphological changes, aberrant cellular differentiation, genetic damage and molecular pathways responsible for premalignant changes and carcinogenesis in each compartment. Specifically, we are currently working on premalignant models for adenocarcinoma and small cell lung cancer. A. Premalignant changes of lung adenocarcinoma We have previously shown that field cancerization in human lung is associated with alterations in the expression patterns of both peripheral airway cell and neuroendocrine markers. We are now developing experimental models in which the pulmonary changes parallel those seen in man. We have previously shown that a neural transcription factor from Drosophila, achaete-schute homolog-1 (hASH1 in humans and Mash1 in mice) is expressed at high levels in neuroendocrine lung cancers and is essential for neuroendocrine differentiation in neoplastic and non-neoplastic lung. In contrast, constitutive expression of the achaete-schute homolog-1 transcription factor under CC10 promoter caused marked bronchialization of alveoli, which provides a model for a potential premalignant lesion for human lung adenocarcinomas. We have previously shown that such lesions occur in 12% of human lung cancer resection specimens being part of the field cancerization phenomenon. Using our mouse model we have now demonstrated that the lesions are due to sustained cellular proliferation and resistance to apoptosis in terminal bronchioles involving several pathways known to be activated during carcinogenesis. Moreover, we recently discovered that matrilysin-1, also called matrix metalloproteinase-7, was expressed in both human and mouse lesions of bronchiolization of alveoli, but not in normal lungs. In vitro, forced expression of the matrilysin-1 gene in immortalized human airway cells promoted cellular migration, proliferation and protection from apoptosis. Gene expression analysis uncovered several genes that were also related to cell growth, migration and death. In vivo, the bronchiolization of alveoli was reduced in mice that were deficient of matrilysin-1. We conclude that matrilysin-1 may play an important role in this candidate premalignant lesion by promoting proliferation, migration and attenuation of apoptosis and involving multiple genes in the MAP kinase pathway. We are currently assessing further the specific molecular mechanisms involved in cellular migration that are potentially regulated by hASH1 in cells derived both from airways and pulmonary carcinomas. The significance of our findings is two-fold: First, we show that constitutive expression of hASH1 in lung epithelium promotes remodeling through the same pathways that are commonly activated during lung carcinogenesis thus providing a novel model for the potential premalignant lesion of human lung adenocarcinomas. Second, our results suggest that the impact of hASH1 is not limited to neuroencocrine cells, but is extended to epithelial (non-neuroendocrine) cells as well. B. Neuroendocrine differentiation and cellular lineages in the lung during carcinogenesis. Exposure to naphthalene, a component of cigarette smoke, kills lung airway epithelial (Clara) cells in mice, but is rapidly followed by Clara cell reconstitution coincident with proliferation of pulmonary neuroendocrine cells. The reconstituting progenitor cells have been suggested to enter a transient neuroendocrine differentiation phase before differentiating to Clara cells. Furthermore, these progenitors were suggested to be the target population for transformation to the most common and virulent of all neuroendocrine tumors: small cell lung cancer (SCLC) whose precursor lesions remain unknown. To determine the relevance of the neuroendocrine phenotype to post-naphthalene reconstitution, we examined the role of Protein Gene Product 9.5 (PGP9.5) which is known to be highly expressed in nervous system. Recently, PGP9.5 has also been found to be upregulated in the pulmonary epithelium of smokers and non-small cell lung cancer (NSCLC). Interestingly, the expression of PGP9.5 was transiently upregulated in the non-neuroendocrine airway epithelium in the days immediately following napththalene injection as indicated by immunohistochemistry and mRNA levels. This was associated with transient downregulation of the cell cycle regulator p27(Kip1) previously linked with PGP9.5 in lung cancer cells. This study is the first to specifically identify the carcinogen napthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after lung injury. Our findings strengthen the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis. Moreover, the results suggest that there is a close relationship with neuroendocrine and epithelial phenotypes in the airways during injury repair, which warrants further investigation. We are currently conducting experiments to determine if constitutive expression of PGP9.5 will modify the roles of neuroendocrine and Clara cells during extended repair to injury or carcinogenesis utilizing transgenic mice. It is currently thought that pulmonary neuroendocrine cells, like their counterparts in the gastrointestinal tract are derived from multipotent progenitors, not from neural crest and that all epithelial cells arise from a single cell. However, pulmonary neuroendocrine cells reveal a close relationship with non-neuroendocrine cells, which manifests as co-expression of neuroendocrine markers such as CGRP, PGP9.5 or synaptophysin and epithelial markers such as CC10 and surfactant proteins in the airway epithelium during embryonal development, injury repair and carcinogen exposure. We have previously shown that achaete-schute homolog-1 (Mash1) is essential for the formation of lung neuroendocrine cells. As the relative number of these cells peaks at the time of birth, it is plausible that prior to birth Mash1-defined progenitors may primarily contribute to the generation of neuroendocrine cells. On the other hand, we postulate that Mash1-expressing precursors will participate in epithelial expansion at times of significant growth and maturation of non-neuroendocrine epithelium which takes place immediately following birth and again at the time of injury repair. As indicated above, in humans repeated injury and repair in smokers lungs is an integral part of carcinogenesis. We have now initiated in vivo lineage studies of Mash1-containing cells in the lungs using a dynamic approach utilizing novel genetically modified mouse models that have become available through collaboration. So far we have been able to demonstrate that, similarly to brain and spinal cord, Mash1- derived progenitors in the lung give rise to multiple cell types. We are currently in the process of determining the differentiation patterns of the cells utilizing neuroendocrine and epithelial (non-endocrine) markers. The significance of our research is that the results will provide a rational basis for early detection and novel molecular ta [summary truncated at 7800 characters]